Protein arginine (arg) methylation is a common posttranslational modification of RNA binding proteins (RNA BPs) in yeast and mammals. In Trypanosoma brucei, gene regulation is largely effected posttranscriptionally, and thus likely involves a plethora of RNA BPs. For these reasons, we hypothesize that RNA BP arg methylation plays an especially important role trypanosome gene regulation. Previously, we showed that the mitochondrial RNA BP, RBP16, is an in vivo substrate of the protein arg methyltransferase (PRMT), TbPRMT1. Arg methylation increases the functional diversity of RBP16 and modulates its macromolecular interactions. Additionally, depletion of either TbPRMT1 or TbPRMT5 results in distinct defects in mitochondrial gene expression. We identified several additional RNA BPs and RNA metabolic enzymes that associate with T. brucei PRMTs. One of these is TbDed1, a predicted RNA helicase that is methylated by TbPRMT5, exhibits ATPase activity, and is essential for optimal procyclic form (PF) growth. Ongoing in vivo and in vitro analyses of the five TbPRMTs revealed that TbPRMT6 is essential for optimal PF growth, and TbPRMT7 is a novel kinetoplastid-specific type III PRMT. Our long-term goal is to understand the roles of protein arg methylation in T. brucei gene regulation, and here we propose four specific aims. In Aim 1, we will use in vitro methylation assays and RNAi to complete the comprehensive assessment of T. brucei's capacity for protein arg methylation and evaluate the consequences of PRMT depletion for trypanosome growth and morphology. We will also address redundancy and cooperation of selected TbPRMT pairs by dual RNAi. In Aim 2, a battery of novel methylproteins will be identified by anti-methylarg immunoprecipitation/mass spectrometry, and by TbPRMT6-PTP affinity chromatography. In Aim 3, we will analyze the mechanisms by which arg methylation impacts mitochondrial gene expression. Using cells expressing distinct hypomethylated versions of the model methylprotein, RBP16, we will elucidate the roles of specific methylargs in mRNA stability and editing, higher order complex formation, and mRNA association. Proteins that read RBP16 methylmarks will be identified. We will isolate additional mitochondrial methylproteins, implicated by our previous studies, by anti-methylarg immunoprecipitation. In Aim 4, we will determine the role of arg methylation in creating functional diversity in the DEAD box protein, TbDed1. Cells either depleted of TbDed1 or expressing predominantly hypomethylated TbDed1 will be assayed for gene regulatory functions including trans splicing, translation initiation, and the formation of cytoplasmic RNP granules implicated in RNA turnover and translational control. We will also determine the impact of methylation on TbDed1 ATPase, RNA helicase, and RNA annealing activities to address the molecular bases of cellular methylarg function. Collectively, the proposed studies will significantly increase our understanding of gene regulation in a medically and economically important parasite. They will also provide insight into novel functions of a common posttranslational modification in higher eukaryotes.